Calretinin and calbindin architecture of the midline thalamus associated with prefrontal-hippocampal circuitry.

The midline thalamus bidirectionally connects the medial prefrontal cortex (mPFC) and hippocampus (HC) creating a unique cortico-thalamo-cortical circuit fundamental to memory and executive function. While the anatomical connectivity of midline thalamus has been thoroughly investigated, little is known about its cellular organization within each nucleus. Here we used immunohistological techniques to examine cellular distributions in the midline thalamus based on the calcium binding proteins parvalbumin (PV), calretinin (CR), and calbindin (CB). We also examined these calcium binding proteins in a population of reuniens cells known to project to both mPFC and HC using a dual fluorescence retrograde adenoassociated virus-based tracing approach. These dual reuniens mPFC-HC projecting cells, in particular, are thought to be important for synchronizing mPFC and HC activity. First, we confirmed the absence of PV+ neurons in the midline thalamus. Second, we found a common pattern of CR+ and CB+ cells throughout midline thalamus with CR+ cells running along the nearby third ventricle (3V) and penetrating the midline. CB+ cells were consistently more lateral and toward the middle of the dorsal-ventral extent of the midline thalamus. Notably, single-labeled CR+ and CB+ zones were partially overlapping and included dual-labeled CR+ /CB+ cells. Within RE, we also observed a CR and CB subzone specific diversity. Interestingly, dual mPFC-HC projecting neurons in RE expressed none of the calcium binding proteins examined, but were contained in nests of CR+ and CB+ cells. Overall, the midline thalamus was well organized into CR+ and CB+ rich zones distributed throughout the region, with dual mPFC-HC projecting cells in reuniens representing a unique cell population. These results provide a cytoarchitectural organization in the midline thalamus based on calcium binding protein expression, and set the stage for future cell-type specific interrogations of the functional role of these different cell populations in mPFC-HC interactions.

Inactivation of nucleus reuniens impairs spatial working memory and behavioral flexibility in the rat.

The hippocampal formation (HF) and medial prefrontal cortex (mPFC) play critical roles in spatial working memory (SWM). The nucleus reuniens (RE) of the ventral midline thalamus is an important anatomical link between the HF and mPFC, and as such is crucially involved in SWM functions that recruit both structures. Little is known, however, regarding the role of RE in other behaviors mediated by this circuit. In the present study, we examined the role of RE in spatial working memory and executive functioning following reversible inactivation of RE with either muscimol or procaine. Rats were implanted with an indwelling cannula targeting RE and trained in a delayed nonmatch to sample spatial alternation T-maze task. For the task, sample and choice runs were separated by moderate or long delays (30, 60, and 120 s). Following asymptotic performance, rats were tested following infusions of drug or vehicle. Muscimol infused into RE impaired SWM at all delays, whereby procaine only impaired performance at the longest delays. Furthermore, RE inactivation with muscimol produced a failure in win-shift strategy as well as severe spatial perseveration, whereby rats persistently made re-entries into incorrect arms during correction trials, despite the absence of reward. This demonstrated marked changes in behavioral flexibility and response strategy. These results strengthen the role of nucleus reuniens as a pivotal link between hippocampus and prefrontal cortex in cognitive and executive functions and suggest that nucleus reuniens may be a potential target in the treatment of CNS disorders such as schizophrenia, attention deficit hyperactivity disorder, addiction, and obsessive-compulsive disorder, whose symptoms are defined by hippocampal-prefrontal dysfunctions.

A PER3 Polymorphism Interacts with Sleep Duration to Influence Transient Mood States in Women.

BACKGROUND: Expression of the clock family of genes in the suprachiasmatic nuclei (SCN) regulates the molecular control of circadian timing. Increasing evidence also implicates clock gene activity in the development of mood disorders. In particular, variation in the PER3 clock gene has been shown to influence diurnal preference and sleep homeostasis. However, there is not currently a clear association between PER3 polymorphisms and mood. This is possibly because the PER3 gene has been shown to influence homeostatic sleep drive, rather than circadian timing, and the PER3 gene may be behaviorally relevant only under chronic sleep loss conditions. METHODS: To test the association between PER3 allele status and impaired mood, a total of 205 healthy women were genotyped for PER3 allele status and responded to previously-validated psychological questionnaires surveying self-reported sleep habits (MEQ, PSQI) and mood. Our mood measures included two measures of short-term, transient mood (state anxiety and mood disturbance) and two measures of longer term, ongoing mood (trait anxiety and depressive symptomology). RESULTS: The PER3 genotype distribution was 88 (42.9%) for PER3(4/4), 98 (47.8%) for PER3(4/5), and 19 (9.3%) for PER3(5/5). Our sleep duration x genotype interaction analyses showed that, relative to longer allele carriers, PER3(4/4) genotypes were at greater risk for transient psychological effects (mood and state anxiety) when they reported reduced sleep durations. CONCLUSION: Sleep duration plays a critical role in understanding the extent to which PER3 allele status relates to mood states.

Nucleus reuniens transiently synchronizes memory networks at beta frequencies.

Episodic memory-based decision-making requires top-down medial prefrontal cortex and hippocampal interactions. This integrated prefrontal-hippocampal memory state is thought to be organized by synchronized network oscillations and mediated by connectivity with the thalamic nucleus reuniens (RE). Whether and how the RE synchronizes prefrontal-hippocampal networks in memory, however, remains unknown. Here, we recorded local field potentials from the prefrontal-RE-hippocampal network while rats engaged in a nonspatial sequence memory task, thereby isolating memory-related activity from running-related oscillations. We found that synchronous prefrontal-hippocampal beta bursts (15-30 Hz) dominated during memory trials, whereas synchronous theta activity (6-12 Hz) dominated during non-memory-related running. Moreover, RE beta activity appeared first, followed by prefrontal and hippocampal synchronized beta, suggesting that prefrontal-hippocampal beta could be driven by the RE. To test whether the RE is capable of driving prefrontal-hippocampal beta synchrony, we used an optogenetic approach (retroAAV-ChR2). RE activation induced prefrontal-hippocampal beta coherence and reduced theta coherence, matching the observed memory-driven network state in the sequence task. These findings are the first to demonstrate that the RE contributes to memory by driving transient synchronized beta in the prefrontal-hippocampal system, thereby facilitating interactions that underlie memory-based decision-making.

Dual medial prefrontal cortex and hippocampus projecting neurons in the paraventricular nucleus of the thalamus.

The paraventricular nucleus (PVT) of the midline thalamus is a critical higher-order cortico-thalamo-cortical integration site that plays a critical role in various behaviors including reward seeking, cue saliency, and emotional memory. Anatomical studies have shown that PVT projects to both medial prefrontal cortex (mPFC) and hippocampus (HC). However, dual mPFC-HC projecting neurons which could serve a role in synchronizing mPFC and HC activity during PVT-dependent behaviors, have not been explored. Here we used a dual retrograde adenoassociated virus (AAV) tracing approach to characterize the location and proportion of different projection populations that send collaterals to mPFC and/or ventral hippocampus (vHC) in rats. Additionally, we examined the distribution of calcium binding proteins calretinin (CR) and calbindin (CB) with respect to these projection populations in PVT. We found that PVT contains separate populations of cells that project to mPFC, vHC, and those that innervate both regions. Interestingly, dual mPFC-HC projecting cells expressed neither CR nor CB. Topographically, CB+ and CR+ containing cells clustered around dual projecting neurons in PVT. These results are consistent with the features of dual mPFC-vHC projecting cells in the nucleus reuniens (RE) and suggestive of a functional mPFC-PVT-vHC system that may support mPFC-vHC interactions in PVT-dependent behaviors.

Discharge characteristics of neurons of nucleus reuniens across sleep-wake states in the behaving rat.

The nucleus reuniens (RE) of the ventral midline thalamus is strongly reciprocally connected with the hippocampus (HF) and medial prefrontal cortex (PFC), serving a critical role in affective and cognitive functioning. While midline thalamic nuclei have been implicated in the modulation of states of arousal and consciousness, few studies have addressed RE's role in behavioral state control. Accordingly, as a first line of investigation, we examined the discharge properties of RE neurons in behaving rats throughout the sleep-wake cycle. We analyzed 153 units in RE which demonstrated heterogeneity in discharge rates and pattern of activity across sleep wake states. Using a rate ratio of activity in wake vs. REM, we found that the majority of cells displayed state-related changes and were classified into distinct cell types, exhibiting their highest discharge rates during active waking (AW), REM sleep, or maintaining equivalent activity across AW/REM. We further distinguished cells as either slow firing (SF = < 10 Hz) or fast firing (FF =>10 Hz) cells. The majority of cells, independent of state-related preference, were SF. FF RE cells were primarily wake active and wake/REM cell types. This diverse set of RE neurons are likely modulated by key brainstem and hypothalamic nuclei, which in turn, drive RE to exert strong effects on its cortical targets during waking and REM sleep. RE may not only act as a node in HF-PFC circuitry, but also as a critical thalamic link in ascending arousal and attentional networks.

RatHat: A Self-Targeting Printable Brain Implant System.

There has not been a major change in how neuroscientists approach stereotaxic methods in decades. Here, we present a new stereotaxic method that provides an alternative approach to a traditional u-frame stereotaxic device and reduces costs, surgical time, and aids repeatability. The RatHat brain implantation system is a 3D-printable stereotaxic device for rats that is fabricated prior to surgery and fits to the shape of the skull. RatHat builds are directly implanted into the brain without the need for head-leveling or coordinate-mapping during surgery. The RatHat can be used in conjunction with the traditional u-frame stereotaxic device, but does not require the use of a micromanipulator for successful implantations. Each RatHat contains several primary components including the implant for mounting intracranial components, the surgical stencil for targeting drill sites, and the protective cap for preventing damage from impacts and debris. Each component serves a unique function and can be used together or separately. We demonstrate the feasibility of the RatHat in four different proof-of-principle experiments: (1) a three-pole cannula apparatus, (2) an optrode-electrode assembly, (3) a fixed-electrode array, and (4) a tetrode hyperdrive. Implants were successful, durable, and long-lasting (up to nine months). RatHat print files are easily created, can be modified in computer aided design (CAD) software for a variety of applications, and are easily shared, contributing to open science goals and replications. The RatHat has been adapted to multiple experimental paradigms in our lab and should be a useful new way to conduct stereotaxic implant surgeries in rodents.

Differential effects of mild chronic stress on cortisol and S-IgA responses to an acute stressor.

While acute stress is adaptive in the short term, chronic stress may interfere with HPA axis functioning and self-regulation that can, in turn, alter the body's immune response. Several studies suggest that acute and chronic stress consistently increase cortisol levels; however, the same cannot be said about S-IgA levels. We tested the effects of a mild chronic stress (an academic exam period), on cortisol and S-IgA responses to an acute stress. Exposure to an acute stressor significantly increases cortisol levels during periods of no-stress, but not during mild chronic stress, while S-IgA levels consistently increase during both sessions. Furthermore, we find that during the period of chronic mild stress, the perception of stress is related to increased cortisol response to an acute stressor. Combined, these findings shed light on the impact of increased background stress on acute stress responses.